Research Proposal on REMOVAL OF

PHENOLIC COMPOUNDS
FROM OIL REFINERY WASTEWATER BY
MUTATED BACTERIA, GORDONIA SPUTI
1
S M Nazmuz Sakib (Orchid- https://orcid.org/0000-0001-9310-3014) (sakibpedia@gmail.com)

1
Author Biography

S M Nazmuz Sakib is an eLearning expert and done more than 500 MOOCs or Massive Open Online Courses and experienced as an
instructor in sites like Udemy. He has completed his BSc in Business Studies from School of Business And Trade, Switzerland with CGPA 4 in
the scale of 4 and 97.06% grade marks on an average. He is also a certified Google IT Support Professional, Google Data Analytics Professional
and IBM Customer Engagement Specialist Professional.

1 1
Graduate of BSc in Business Studies Student of LLB(Hon’s)
School of Business And Trade Department of Law
Pilatusstrasse 6003, 6003 Luzern, Dhaka International University
Switzerland House # 4, Road # 1, Block - F, Dhaka 1213
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Student of BSc in Civil Engineering Student of BSc in Physiotherapy
Department of Civil Engineering Faculty of Medicine
Sonargaon University University of Dhaka
147/I, Green Road, Panthapath, Dhaka Nilkhet Rd, Dhaka 1000
Abstract
Water is the need of abiding living beings. Just modest quantity of water is accessible for eating
reason and in this limited quantity an assortment of the water is go through in considerations as
contaminated. As the human games exercises are fostering every day so a report outfitted with
the asset of the utilization of UNO, steady with this situation dramatic expansion in
overall populace is setting weight on the restricted gleaming water property as they're
diminishing . In this 21st century, because of urbanization and industrialization water our bodies
have become contaminated. Likewise with the expansion in industrialization our earth is
uniquely dirtied and, in this contamination, most extreme imperative trademark is finished with
the asset of the use of fragrant poisons. Wastewater coming from enterprises or metropolitan
authority incorporate dirtied substances that might cause horrendous outcomes. Drinking
wastewater can case various Gastrointestinal track issues. Wastewater inconvenience up push up
as a stop outcome simultaneously as untreated water is release into the water our bodies through
agrarian, city, and modern games exercises . Untreated wastewater now not excellent reason
horrible human outcomes but rather it might besides harms the climate and oceanic life .
Untreated wastewater grade by grade impacts the science of water and soil.
Keywords: Biotechnology, Safe water, Water pollution, Environmental Science, Environmental
Chemistry, Waste water

Chapter 1 Introduction:

Small amount of water is available for drinking purpose and in this small amount much of the
water is consider as polluted. As the human activities are increasing day by day so a report
presented by UNO, according to this scenario exponential increase in world population is
putting stress on the limited fresh water resources as they are decreasing (Azizullah et al. 2011).
In this 21st century, due to urbanization and industrialization water bodies are becoming
polluted. As with the increase in industrialization our earth is highly polluted and, in this
pollution, main role is played by aromatic pollutants. Wastewater coming from industries or
municipal authority contain polluted substances that can cause adverse effects. Drinking
wastewater can case a lot of Gastrointestinal track problems. Wastewater problem arise as a
result when untreated water is discharge into the water bodies through agricultural, municipal,
and industrial activities (Arafath et al. 2019). Untreated wastewater not only cause adverse
human effects but it can also damages the environment and aquatic life (Spina et al. 2012).
Untreated wastewater gradually affects the chemistry of water and soil and biodiversity because
it produces serious hazardous gasses (Shukla et al. 2020).

1.1) Industrial wastewater

With the rapid need of mankind to improve its life style, led to the enormous increase in
domestic and industrial actions as result of this large amount of untreated pollutants that are
present in industrial wastewater are discharge into the waterbodies (Anku et al. 2017). Industrial
wastewater include wastewater coming from all industries i.e. textile, petroleum, distilleries,
leather, pharmaceutical, agrochemical industry, steel, rubber, and all industries around the globe.
Wastewater generating from industries contain multi components. Industries are releasing
enormous amount of toxic, nontoxic, aromatic, organic, inorganic and undegradable pollutant
into the environment. Waterborne diseases are cause by the industrialization and they are
responsible in the death rate of people up to 3.4 million each year.

In petroleum refinery (Fadhil and Al-Baldawi 2020), to fulfill the daily energy requirement 97
million oil barrels were required. In underdeveloped countries, due to accidental damage during
transportation (shipping) of oil, may release oil on to the land or the wastewater coming from
these refineries may not be treated and directly disposed into the oceans. This polluted water
may cause serious health effects and it affects the marine flora and fauna.

Textile industry (Balapure et al. 2016) is the backbone of every country and it produce
large amount of liquefied and undegradable pollutants. Pollutants from textile comes in the form
of dye that require for staining cotton. In dyeing process, about 80,000 tons/year dye is being
used which contain large amount of heavy metals and this dye containing wastewater is
discharge into the fields or in the aquatic system and affects food chains.

Pharmaceutical industries (Rana et al. 2017) are responsible for producing antibiotics,
drugs, steroid, analgesics, heart stimulants etc. to protect the mankind. The process involves by
these industries in producing these products may produces some serious effluents, so the
wastewater coming from this industry contain color, has some toxic odor and may evolve
poisonous gasses which affects the mankind.
 1.2) Phenolic contaminants:
Among pollutants aromatic phenol is in the highlight. Phenol (Al-Defiery and Reddy
2019) is an aromatic compound in which hydroxyl group is attached to the benzene ring. It has a
sweet odor. Its chemical formula is C6H5OH. Phenol and all its related classes that are present in
the wastewater are produced by industries and are considered as highly toxic and poisonous.
Phenol is highly toxic as 50ppb of phenol is harmful to aquatic life as well as 1g phenol is toxic
to human beings. Phenol release from various industries contaminate the natural ecosystem like
soil, air and water. It is considered as harmful to skin; mucus lining and eyes and it may cause
intense burning and irritation and may cause gastrointestinal track disorders.

Phenol is also termed as carbolic acid. European Union and United States Environmental
Protection Agency regarded phenol as toxic or extremely significant pollutant. Phenols can be
classified according to the nature and number of benzene ring (phenol ring) and the group attach
to them as meta, para and ortho phenol. Phenols that are present in the water comes from two
sources man-made (anthropogenic) and natural. Natural source of phenol present in water
include the dead organic corpse of animals and plants. Whereas man-made sources include
industries, agricultural activities, the wastewater discharge from industries contain high level of
phenol.(Anku et al. 2017)

Among all the organic pollutants, phenol is the most priority effluent present in
wastewater coming from industries. Industries that are in highlight in discharging phenol in
wastewater include textile, pharmaceutical, petroleum refineries, leather, chemical, paper
industries. Due to accidental discharge of oil during shipping a report conducted on aquatic life
about 1.5mg/l phenol is highly lethal to carp and 0.26mg/l phenol is toxic to shrimps. As a
result, phenol is not only hazardous to the human being but also to the aquatic life (Li et al.

2019).

1.3) Treatment strategy through bioremediation:

These effluents that are discharge form the industries badly affects the ecosystem, so protective
strategies that involve degradation of these organic effluents were used (Iqbal et al. 2018).
Bioremediation is a process in which living organisms are used to control/degrade the non-living
substances. Bioremediation is a biological control and ecofriendly strategy of pollutants
controlling. Bioremediation is used for the recycling of wastewater and for the removal of heavy
chemicals. In bioremediation microorganisms utilize pollutants as a source of carbon and
energy.

Bioremediation take place in 3 different strategies; the microorganisms use in this

process either completely transform that particular chemical or it may cause the disintegration of
molecule or may convert it into simpler nontoxic form like CO 2 and H2O. Extensive degradation
properties played by microorganisms , bioremediation involves the use bacteria, fungi, algae
(Krastanov et al. 2013). Wastewater is highly polluted and contain large amount of toxic
chemicals like phenol, pesticides etc. so direct addition of microorganisms to wastewater may
inhibit the process and as a result microorganism may die and the process stop because microbes
can’t tolerate the high concentration of phenol because of its toxicity. So, for the effective
recycling of wastewater through bioremediation, pretreatment is required for the wastewater. As
pretreatment will decrease the level of toxicity (Bajaj et al. 2008).

Bioremediation is extensively used for treating the water contaminated with industrial
waste i.e. phenols and other pollutants, soil contaminated with diesel and other petroleum
products (Poi et al. 2017). In bioremediation highly active strains of microorganisms are utilized
to degrade recalcitrant pollutants. Remediation strategy (Rana et al. 2017) is of two types
aerobic and anerobic depending upon the type and nature of microorganisms. Anerobic
bioremediation is used when anerobic bacteria involved and aerobic bioremediation is used
when aerobic bacteria is used.

1.4) Factors that affects the Biodegradation of phenol:

To carry out bioremediation efficiently there are following factors which are needed to
be control. These factors are temperature, PH, nutrition availability, oxygen content (Singh et al.
2020).

a. Temperature: Every bacterium works at an optimum temperature. Temperature affects

the activity of the enzymes that are involve in biodegradation. Increase or decrease in
temperature cause the biodegradation process to stop. Optimum temperature for most of
the bacteria is 30 ℃.
 b. PH: PH is also an important factor that is involve in the biodegradation process. Most of
the microorganisms can’t tolerate the PH more than 9 or less than 4. So as a result,
bacteria neither tolerate extreme acidic nor extreme basic.
c. Nutrition Availability: Different media are used to provide nutrition to the bacteria. The
most important nutrition requirement of most of the bacteria is Carbon, Nitrogen and
Phosphorus.
d. Oxygen Content: Both bacteria are use in the biodegradation process aerobic and
anerobic bacteria. But most of the biodegrading process are carryout with aerobic
bacteria as this bacterium require oxygen and are easy to culture. Oxygen requirement of
this bacteria is 21%. Whereas anaerobes do not require oxygen and they have many
environmental drawbacks.
e. Substrate availability: when large amount of substrate is present microbes will replicate
at the faster rate to degrade that substrate. When the biodegradation process will stop, as
all the substrate is degraded microbial population will start to decline because no food is
available for microbes.

1.5) Bacterial Characteristics to remove phenolic compounds:

High concentration of phenol is present in the wastewater, this high concentration is
toxic to the microbial specie use for its degradation, as some of the species will tolerate this high
concentration while some of the specie die as a result degradation process will be stopped
(Suhaila et al. 2019). Wastewater discharge form industries contain highly toxic pollutants.
These pollutants are highly reactive so the microbial strain use to degrade these effluents must
be strong, that it must not be affected by these pollutants (Santos and Linardi 2004).

Bacteria is highly effective and most widely used to degrade the phenol present in wastewater.
The most commonly use media for the growth of bacteria is Minimal Salt Media (MSM) and
Luria Bertani media (LB media). For biodegradation, bacteria are added to the reaction that run
for the particular time. Bacteria utilize/degrade this phenol in different ways i.e. it uses is as its
energy or carbon requirement or convert it into simpler form. Bacteria degrade the phenol
through ortho or meta degrading pathways. Bacteria can tolerate different range of phenol from
very low i.e. 100mg/l to very high 1200mg/l. Bacteria contain different type of enzymes for
example phenol hydroxylase, catechol dioxygenase, cis, cis-muconate cyclase that are involve
in the biodegradation of phenol (Krastanov et al. 2013)
 Pseudomonas putida can degrade the high concentration of phenol up to 1000mg/l. It
efficiently cleans the wastewater by eliminating non- phenolic and phenolic effluents present in
waste water (Gonzalez et al. 2001). Achromobacter sp. can degrade low concentration of phenol
i.e. 300mg/l in wastewater and at high concentration i.e. 600mg/l Achromobacter degrade and as
a result process stop. Achromobacter sp. not only degrade the phenolic contaminants but it also
degrade the para and meta cresol present in the industrial wastewater
(Gracioso et al. 2019) . As the some of the bacteria can’t tolerate the high concentration of
phenol due to its toxicity and are unable to degrade it. So, to overcome this problem bacterial
strain Pseudochrobactrum sp. mutated by UV radiation was used. This UV mutated bacterial
strain can disintegrate high level of phenol up to 1800mg/l and is resistant to the toxicity of
phenol in wastewater (Mao et al. 2015)

1.6) Fungal characteristics to remove the phenol:

Fungus is also used to degrade the phenol present in industrial water. Different strain of
fungus are use like A.niger, A.fumigatus, penicillium sp. these strain are more commonly use.
The media use by fungus is MSM. These strains have the capacity to bear the toxicity of phenol
upto 10 mM. And when mutated strain is use the degradation ability of that strain increase by
several times as compare to the wild type. Fungus also contain different type of enzymes that
are involve in the biodegradation like phenol hydroxylase, catechol 1,2dioxygense (Santos and
Linardi 2004) (IBRAHIM and AL-GHAMDI 2019).

Chapter 2 Study Objective:

• The purpose of this project is to remove the effluents present in the industrial
wastewater.
• Biological treatment is use because it does not evolve any environmental toxic
substance.
• Bacteria is easy to culture.
• The bacteria use will convert the toxic substance in wastewater into non-toxic form
Chapter3 Material and methods:

3.1) Sample collection

Sample of wastewater will be obtained from the oil refinery. This wastewater contain
phenol. The level of phenol will be check by spectrophotometrically method. The wastewater
then is pre-treated with chemical to neutralize its PH and toxicity.

3.2) Strain isolation

Aerobic bacterial strain Gordonia sputi will be identify by 16S RNA sequencing and
isolated from hydrocarbon contaminated soil. This wild strain will then be mutated by UV rays
to increase its efficiency for degradation.

3.3) Inoculum preparation

Bacteria will grow on Luria Bertani (LB) media. This media will provide all the nutrients
necessary for the bacterial growth.

3.4) Biodegradation experiment:

For the biodegradation of phenol, wastewater sample will add into the batch bioreactor.
After that bacteria culture will be added. And the bioreactor is run for the specific time period.
Temperature, PH, oxygen content will be adjusted so the bacteria will efficiently degrade the
phenol or convert it into non-toxic form. Continuous stirring will be required. The sample will
be drawn periodically to assess the phenol concertation.

3.5) Quality Assessment:

The water obtain after treatment will be treat with activated carbon. Activated carbon
absorb all the remaining impurities present in wastewater. It further removes the unnecessary
color of chemicals from the wastewater. The quality of wastewater will be check by mass
spectrometry method.

Chapter 4 Expected outcome

The expected outcome of this project will be the bacterial strain use will efficiently
degrade the phenol. As the water is pre-treated so most of its toxicity will be eliminated. This
bacterial strain significantly degrades phenol and non-phenolic substances present in
 wastewater. Hence, mutated strain of Gordonia sputi will be a potential candidate involve in the
degradation of phenol Now the water available after treatment will be ready for industrial re-use
or if it is disposed of into river then it will not affect the environment and aquatic life.

Chapter 5 Research timeline

The time period require for this work will be 1 year in a duration form October 2022 to
September 2023.

Activities OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP

Problem
Identification
literature
Review
Synopsis

Sample
collection
Experimental
Result
Statistical
Analysis
Research
paper
Thesis
Viva

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